During pregnancy in both rodents and humans, natural killer (NK)-lineage cells accumulate in large numbers in the maternal tissue (decidua) at the implant site, then in a unique structure in rodents, the metrial gland. These cells, termed granulated metrial gland (GMG) cells, have many characteristics of highly lytic IL-2-activated NK cells (e.g., large size and granules with lytic proteins), yet they have poor lytic capacity in vitro towards prototype targets. These cells arise rapidly through proliferation and differentiation from precursors in intimate association with decidual cells, but how the decidual cells influence these events is not known. The long-term goal of our research is to understand how these cells develop and function in pregnancy. Toward this end, the proposed studies are focussed on elucidating the features of the decidual microenvironment in rodents that regulate the development of these cells along a particular phenotypic and functional pathway. In Aim 1, decidual tissue, taken during the time of NK cell differentiation, and metrial gland tissue will be surveyed for the presence of cytokines which can influence NK cells, using ELISA assays and reverse transcription- polymerase chain reaction. Those identified will be localized to determine if they are produced by the decidual cells or by the differentiating NK cells themselves. Aim 2 involves characterization of rat GMG cells with regard to specific markers, lectin binding, and receptor expression, which will further define these cells in relation to classically activated NK populations and provide other markers to monitor during in vitro studies. In Aim 3, two in vitro approaches will be taken to elucidate the factors in decidua that direct proliferation and differentiation of NK cells into the GMG morphological and functional phenotype. First, splenic NK cells and bone marrow progenitor cells will be incubated with the cytokines identified in decidual cells, along with prostaglandins, decidual prolactin-like proteins, and ECM components to determine the combination that induces differentiation of GMG-type cells. Second, NK cells or progenitor cells will be co-cultured with purified decidual cells to establish a model for GMG cell development, then antagonists such as neutralizing antibodies will be used to determine the factors relevant to GMG cell differentiation. In Aim 4, nonpregnant and pregnant animals will be infused with viable fluorescent-labelled precursor cells and endometrium, decidua, and metrial gland tissues subsequently assessed by fluorescence microscopy to determine the time course of GMG precursor cell immigration, using antibodies to identify labelled cells entering these sites. Although their function is unknown, the striking presence of GMG cells in the pregnant uterus suggests that they play some important role in pregnancy. Furthermore, NK-lineage cells are strongly implicated in certain cases of pregnancy failure, suggesting that alterations in the normal regulation of decidual NK cell differentiation may have adverse effects on pregnancy. The proposed studies will provide significant new information towards understanding the development of these cells.